The mechanisms involved in the metabolic activation and toxicity of 2-methylfuran (2-MF), a naturally occurring cytotoxic furan found in cigarette smoke and coffee are being investigated. 2-MF and 3-methylfuran are bioactivated in vitro by microsomal mixed function oxidases to acetylacrolein and methylbutenedial, respectively, that bind covalently to microsomal protein. Unsaturated aldehydes can react with both protein and DNA either via Michael addition across the activated double bond or nucleophilic addition to the aldehyde. Following administration of 2-[(14C)methyl]furan to rats, extensive covalent binding of label to macromolecules in liver, lungs and kidney was observed. Smaller amounts were bound to tissues with little or no known mixed function oxidase activity. Maximal covalent binding to both protein and DNA was observed in the liver, the target organ where toxicity is manifested. Liver GSH levels decreased by a third, half an hour after administration of 2-MF indicating the formation of electrophilic metabolites. Pretreatment with various inhibitors and inducers of metabolism showed that phenobarbital potentiated toxicity of 2-MF and was followed by increased urinary excretion of label, while 3-methylcholanthrene or piperonylbutoxide did not markedly alter the toxicity of 2-MF. Pretreatment with buthionine sulfoximine (BSO), a GSH depleter and a chemosensitizing agent, while decreasing covalent binding, also decreased toxicity, whereas diethylmaleate, also a GSH depletor, increased both covalent binding and toxicity of 2-MF. BSO, which depletes GSH levels by inhibiting cysteine systhetase is known to enhancae cysteine levels in tissues. Thus, BSO probably decreases covalent binding by trapping the reactive intermediate as the stable systeine conjugate.